The precise control of blood glucose levels is of vital importance to humans as well as other mammals. It is well established that the two hormones insulin and glucagon are important for maintenance of correct blood glucose levels. While insulin acts in the liver and peripheral tissues by reducing blood glucose levels via increased peripheral uptake of glucose and reduced glucose output from the liver, glucagon acts mainly on the pancreas and liver, by increasing blood glucose levels via up-regulation of gluconeogenesis and glycogenolysis. Glucagon has also been reported to increase lipolysis, to induce ketosis and to reduce plasma triglyceride levels in plasma [Schade and Eaton, Acta Diabetologica, 1977, 14, 62].
Glucagon is an important part of the defense mechanism against hypoglycaemia and administration of a low dose of glucagon may prevent insulin-induced hypoglycaemia or improve the ability to recover from hypoglycaemia. Glucagon agonism has also been shown to exert effects on lipid metabolism, energy expenditure and food intake [Habegger et al. Nature Reviews Endocrinology 2010, 6, 689-697].
A large number of people suffering from diabetes, in particular Type 2 diabetes, are over-weight or obese. Obesity represents a high risk factor in serious and even fatal common diseases and for most diabetics it is highly desirable that their treatment does not cause weight gain.
Several patent applications disclosing different glucagon-based analogues and GLP-1/glucagon receptor co-agonists are known in the art, such as e.g. patents WO2008/086086, WO2008/101017, WO2007/056362, WO2008/152403 and WO96/29342. Other glucagon analogs disclosed are PEGylated (e.g. WO2007/056362) or acylated in specific positions of native human glucagon (e.g. WO96/29342). Glucagon peptides for prevention of hypoglycaemia have been disclosed, as e.g. in U.S. Pat. No. 7,314,859.
Glucagon is of limited potential use in pharmaceuticals due to fast clearance from circulation with a half life of approximately 5 min. A high clearance of a therapeutic agent is inconvenient in cases where it is desired to maintain a high blood level thereof over a prolonged period of time, since repeated administrations will then be necessary. In some cases it is possible to influence the release profile of peptides by applying suitable pharmaceutical compositions, but this approach has various shortcomings and is not generally applicable.
Glucagon is currently available as a freeze-dried formulation, with a short duration of action, restricted to less than an hour in spite of a glucagon level that peaks at levels far higher than endogenous glucagon levels. There is therefore a need for chemically modified glucagon compounds in order to be delivered at continuous levels, so that longer biological half-life is achieved, i.e. modified glucagon peptides with a protracted profile of action.
The physical as well as the chemical stability of glucagon is poor when dissolved in an aqueous solution. Solutions of glucagon form gels and fibrils within hours or days, depending on purity of the peptide, salt concentration, pH and temperature (Beaven et al. European J. Biochem. 1969, 11, 37-42). Glucagon contains several labile amino acids or amino acid sequences that may give rise to deamidation, cleavage, aspartimide formation and isomerisation. In addition the solubility of human glucagon is very poor in the pH range from 3.5-9.5.